The ability to quantify acute, non-steady state changes in substrate dynamics is imperative in the study of in vivo regulation of metabolism. Isotopic tracer techniques have been devised for this purpose. The most common approach is a single-pool model, known to be based on incorrect assumptions when applied to glucose kinetics, which has been demonstrated in several studies to be unreliable in rapidly changing situations. Two- pool models have also been described, but not widely accepted. Our hypothesis is that current methods for the calculation of non-steady plasm, a glucose kinetics can be improved by two innovations: (1). Basing model development on the measurement of glucose concentration and enrichment and insulin concentration in the two major compartments in which they are distributed: plasma and interstitial fluid. Interstitial fluid measurements will be obtained by sampling from a cannula chronically placed in the thoracic duct of sheep. (2). Data analysis will involve the so- called "direct" approach, which makes use of the governing differential equations, thereby allowing prescribed precision of statistical estimation. In order to assess the applicability of this approach, experiments will be performed in conscious sheep and in normal human volunteers. Experiments will be performed in the setting of either constant or varying insulin and glucose concentrations. In the later cases, the equations will account for the relationship between the values for the rate parameters and the prevailing insulin and glucose concentrations. Whereas the tests of this new approach to modeling will be focused on glucose, it is not restrictive and we anticipate that subsequent to these validation experiments the approach will become widely applied to the study of a variety of compounds.